Biology Reference
In-Depth Information
Chapter 6
Synthesis and Validation of Cyanine-Based Dyes for DIGE
Michael E. Jung , Wan-Joong Kim , Nuraly K. Avliyakulov ,
Merve Oztug , and Michael J. Haykinson
Abstract
The application of difference gel electrophoresis (DIGE), in particular its most common “minimal labeling”
variety, utilizes N -hydroxysuccinimide esters of Cy2, Cy3, and Cy5 dyes, which are commercially available.
We describe methods for the effi cient synthesis of all three dyes from relatively inexpensive and commercially
available precursors in only a few steps and with relatively high yields. In model DIGE experiments, the
newly synthesized dyes proved to be indistinguishable from commercially available ones and have been
shown to be stable for years while stored under argon as dry solids or after being dissolved in N ,
N -dimethylformamide.
Key words: DIGE, Difference gel electrophoresis, Benzoxazolium Cy2, Propyl Cy3, Ethyl Cy5,
Cy dye synthesis
1. Introduction
Difference gel electrophoresis (DIGE) is the latest in a series of
major advances in the fi eld of two-dimensional (2D) gel electro-
phoresis, which was originally introduced by O'Farrel ( 1 ). The key
feature of the DIGE method relies on labeling of protein samples
before electrophoresis with fl uorescent dyes (originally using two
N -hydroxysuccinimide (NHS) esters of Cy3 and Cy5) followed by
protein separation of the combined samples on the same 2D gel
( 2 ). Multiplexed labeled proteins comigrate in the gel ensuing full
coregistration of spectrally different gel images, thus minimizing
gel-to-gel technical variation commonly associated with regular
2D gels. The introduction of a third Cy2-based dye allowed the
implementation of an internal standard for DIGE, which further
improved the method. The internal standard concept is based on
labeling with Cy2 a pooled sample consisting of equal aliquots of
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